Mutations within the p53 gene leading to accumulation of non-functional faulty protein occur in nearly 50% of cancer cases. Loss of p53 function results in abrogation of suicidal programs that produce genetic stability and intrinsic anticancer defense, opening the way for uncontrolled proliferation and malignant progression of cancer cells. Meanwhile, in the cells harboring structural defects within the p53 gene, the other components of the p53 pathway usually remain intact, leaving tumor cells highly sensitive to reintroduced wild-type p53. Theoretically, the impaired activity of mutant p53 protein could be pharmacologically corrected by small molecules, which induce a therapeutic effect. In a preliminary study, a set of small molecules that restore transcriptional activity of the His273 p53 mutant was obtained by high throughput screening of a chemical library in a cell-based readout. Some of the compounds show His273 p53 mutant-dependent growth suppressing and pro-apoptotic activity, and reduced growth of xenografts of A431 cell in nude mice. In Aim 1 of the proposed program, we shall identify small molecules that reactivate transcriptional activity of several classes of p53 mutants in the context of human tumor cells by analyzing results of additional ongoing screenings. These compounds will be classified according to chemical similarity, spectrum of activity toward different classes of p53 mutants, and differences in the mechanisms of action. Detailed characterization of changes within the p53 pathway, caused by the p53-reactivating compounds, and their mechanisms of action will be challenged in Aim 2 of the program. Aim 3 is designed to evaluate therapeutic potentials of restored p53 pathways in human tumor cells bearing mutant p53. Reactivation of p53 pathways will be achieved both by conditional expression of the wild-type p53, and by chemical reactivators of mutant p53 obtained in the course of the project. Optimal combinations of restored p53 activity with other therapeutic treatments will be found to ensure prevalence of cytotoxicity over cytostatic effects. These studies will be conducted both in vitro and in tumor xenografts. Approaches for modulation of the p53-dependnet response in human tumor cells will be developed and experimentally tested. Upon the completion of the project, novel potential classes of anticancer drugs that convert mutant p53 into a therapeutic will be suggested, and therapeutic potentials of restored mutant p53 will be evaluated.